Rho associated protein kinases (ROCKs) are Ser/Thr protein kinases, activated by small GTPases of the Rho family that act as molecular switches to mediate cell signaling. The Rho/ROCK signaling pathway is known to participate in the regulation of numerous cellular functions such as actin cytoskeleton organization, contraction, cell adhesion, motility, and morphology, proliferation, cytokinesis, gene expression, and angiogenesis.
Two isoforms, ROCK1 and ROCK2, have been identified and they share 65% homology in amino acid sequence and 92% homology in their kinase domains. The two isoforms, although ubiquitously expressed, have been found to possess differential tissue distribution. ROCK1 is expressed in lung, liver, stomach, spleen, kidney and testis, whereas ROCK2 is highly expressed in brain, heart and muscle tissues (Nakagawa, et al., “ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice,” FEBS Lett, 1996, 392:189-193). Despite the differential tissue distribution, little is known about the functional differences between the two ROCK isoforms (Sapet, et al., “Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2,” Blood, 2006, 108:1868-1876; Chang, et al., “Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis,” Proc Natl Acad Sci USA, 2006, 103:14495-14500; Sebbagh, et al., “Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing,” Nat Cell Biol, 2001, 3:346-352; Thumkeo, et al., “Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death,” Mol Cell Biol, 2003, 23:5043-55; Shimizu, et al., “ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles,” J Cell Biol, 2005, 168:941-53; Zhang, et al., “Targeted deletion of ROCK1 protects the heart against pressure overload by inhibiting reactive fibrosis,” Faseb J, 2006, 20:916-925; Rikitake, et al., “Decreased Perivascular Fibrosis but Not Cardiac Hypertrophy in ROCK1+/−Haploinsufficient Mice,” Circulation, 2005, 112:2959-2965; Coleman, et al., “Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I,” Nat Cell Biol, 2001, 3:339-45; Sebbagh, et al., “Direct cleavage of ROCK II by granzyme B induces target cell membrane blebbing in a caspase-independent manner,” J Exp Med, 2005, 201:465-471).
ROCKs have been subjected to growing attention, having been implicated in a range of therapeutic areas including cardiovascular diseases (Shimokawa, et al., “Development of Rho-kinase inhibitors for cardiovascular medicine,” Trends Pharmacol Sci, 2007, 28:296-302; Xing, et al., “Rho-kinase as a potential therapeutic target for the treatment of pulmonary hypertension,” Drug News Perspect, 2006, 19:517-522; Liao, et al., “Rho kinase (ROCK) inhibitors,” J Cardiovasc Pharmacol, 2007, 50:17-24; Shimokawa, et al., “Rho-kinase is an important therapeutic target in cardiovascular medicine,” Arterioscler Thromb Vasc Biol, 2005, 25:1767-1775; Dong, et al., “Current status of Rho-associated kinases (ROCKs) in coronary atherosclerosis and vasospasm,” Cardiovasc Hematol Agents Med Chem, 2009, 7:322-330), CNS disorders (Kubo, et al., “Rho-ROCK inhibitors for the treatment of CNS injury,” Recent Pat CNS Drug Discov, 2007, 2:173-9; Kubo, et al., “The therapeutic effects of Rho-ROCK inhibitors on CNS disorders,” Ther Clin Risk Manage, 2008, 4:605-615), inflammation (LoGrasso Philip, et al., “Rho kinase (ROCK) inhibitors and their application to inflammatory disorders,” Curr Top Med Chem, 2009, 9:704-23), and cancer (Suwa, et al., “Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas,” Br J Cancer, 1998, 77:147-152; Kamai, et al., “Overexpression of RhoA, Rac1, and Cdc42 GTPases is associated with progression in testicular cancer,” Clinical Cancer Research, 2004, 10:4799-4805; Schmitz, et al., “Rho GTPases: Signaling, Migration, and Invasion,” Exp Cell Res, 2000, 261:1-12; Imamura, et al., “Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase,”Jpn J Cancer Res, 2000, 91:811-816; Somlyo, et al., “Rho-kinase inhibitor retards migration and in vivo dissemination of human prostate cancer cells,” Biochem Biophys Res Commun, 2000, 269:652-659; Uchida, et al., “The suppression of small GTPase Rho signal transduction pathway inhibits angiogenesis in vitro and in vivo,” Biochem Biophys Res Commun, 2000, 269:633-640; Itoh, et al., “An essential part for Rho-associated kinase in the transcellular invasion of tumor cells,” Nat Med (NY), 1999, 5:221-225; Uehata, et al., “Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension,” Nature, 1997, 389:990-4; Ishizaki, et al., “Pharmacological properties of Y-27632, a specific inhibitor of Rho-associated kinases,” Mol Pharmacol, 2000, 57:976-983; Narumiya, et al., “Use and properties of ROCK-specific inhibitor Y-27632,” Methods Enzymol, 2000, 325:273-84; Nakajima, et al., “Effect of Wf-536, a novel ROCK inhibitor, against metastasis of B16 melanoma,” Cancer Chemother Pharmacol, 2003a, 52:319-24; Nakajima, et al., “Wf-536 prevents tumor metastasis by inhibiting both tumor motility and angiogenic actions,” Eur J Pharmacol, 2003b, 459:113-20; Ying, et al., “The Rho kinase inhibitor fasudil inhibits tumor progression in human and rat tumor models,” Mol Cancer Ther, 2006, 5:2158-2164; Somlyo, et al., “Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants,” Faseb J, 2003, 17:223-234; Hampson, et al., “Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies,” Br J Cancer, 2009, 101:829-839; Igishi, et al., “Enhancement of cisplatin-induced cytotoxicity by ROCK inhibitor through suppression of focal adhesion kinase-independent mechanism in lung carcinoma cells,” Int J Oncol, 2003, 23:1079-1085; Liu, et al., “Inhibition of Rho-Associated Kinase Signaling Prevents Breast Cancer Metastasis to Human Bone,” Cancer Res, 2009, 69:8742-8751; Ogata, et al., “Fasudil inhibits lysophosphatidic acid-induced invasiveness of human ovarian cancer cells,” Int J Gynecol Cancer, 2009, 19:1473-80; Zohrabian, et al., “Rho/ROCK and MAPK signaling pathways are involved in glioblastoma cell migration and proliferation,” Anticancer Res, 2009, 29:119-123).
Co-overexpression of Rho and ROCK proteins in cancer cells has been reported in ovarian cancer, pancreatic, testicular, and bladder cancer (Suwa et al. (1998); Kamai et al. (2004)). Malignant transformation and metastasis require changes in the migratory, invasive and adhesive properties of tumor cells, and changes in the regulation cellular processes depending on the proper assembly/disassembly of actin-cytoskeleton. Each of these events is regulated by Rho/ROCK pathway and plays an important role in the development and progression of cancer (Schmitz et al. (2000)). The implication of Rho/ROCK signalling pathway in invasion by tumor cells (Imamura et al. (2000); Somlyo et al. (2000)), angiogenesis (Uchida et al. (2000)), and their evolution to metastasis (Itoh et al. (1999)) has been amply documented. In light of these findings, the pharmacological inhibition of ROCKs has been suggested as a promising strategy in the prevention of cell invasion, a central event in the process of metastasis (Itoh et al. (1999); Uehata et al. (1997); Ishizaki et al. (2000); Narumiya et al. (2000)).
The potential of ROCK inhibitors as anticancer drugs was demonstrated by the identification of specific ATP competitive inhibitors, Y27632, and Wf536 (FIG. 1) (Itoh et al. (1999); Nakajima et al. (2003a); Nakajima et al. (2003b); Somlyo et al. (2000)), displaying high inhibitory potency for ROCKs. Specifically, Y27632 was reported to reduce metastasis in animal model systems (Itoh et al. (1999)), while Wf-536 has shown efficacy in preventing tumor metastasis in vivo models by inhibiting tumor-induced angiogenesis as well as tumor motility (Nakajima et al. (2003a); Nakajima et al. (2003b); Somlyo et al. (2003)). Han and coworkers have also investigated the ability of Fasudil (5-(1,4-diazepane-1-sulfonyl)isoquinoline) (the only ROCK inhibitor clinically approved in Japan for the treatment of cerebral vasospasm) to inhibit tumor progression in human and rat tumor models (Ying et al. (2006)).
Significant research efforts have been directed towards the identification of more potent and more selective ROCK inhibitors and their use for the treatment of cardiocascular diseases and CNS disorders (Chen, et al., “Chroman-3-amides as potent Rho kinase inhibitors,” Bioorg Med Chem Lett, 2008, 18:6406-6409; Sessions, et al., “Benzimidazole- and benzoxazole-based inhibitors of Rho kinase,” Bioorg Med Chem Lett, 2008, 18:6390-6393; Iwakubo, et al., “Design and synthesis of rho kinase inhibitors (III),” Bioorg Med Chem, 2007, 15:1022-1033; Goodman, et al., “Development of Dihydropyridone Indazole Amides as Selective Rho-Kinase Inhibitors,” J Med Chem, 2007, 50:6-9; Feng, et al., “Discovery of Substituted 4-(Pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as Potent and Highly Selective Rho Kinase (ROCK-II) Inhibitors,” J Med Chem, 2008, 51:6642-6645; Sehon, et al., “Potent, Selective and Orally Bioavailable Dihydropyrimidine Inhibitors of Rho Kinase (ROCK1) as Potential Therapeutic Agents for Cardiovascular Diseases,” J Med Chem, 2008, 51:6631-6634). The antitumor properties of these inhibitors have yet to be shown or published.
The aminothiazole derivative CID5056270 (FIG. 2) has been reported to potently inhibit ROCK2 enzymatic activity with an IC50 values<3 nM (Molecular Libraries Screening Centers Network (MLSCN) (Thomas, et al., “The pilot phase of the NIH chemical genomics center,” Curr Top Med Chem (Sharjah, United Arab Emirates), 2009, 9:1181-1193; Austin, et al., “Policy forum: Molecular biology: NIH molecular libraries initiative,” Science, 2004, 306:1138-1139; Huryn, et al., “The molecular libraries screening center network (MLSCN): identifying chemical probes of biological systems,” Annu Rep Med Chem, 2007, 42:401-416), assay ID 644). CID5056270 displayed a high potency in FRET-based Z′-Lyte biological assay (FIG. 3) (Kang, et al., “Identification of small molecules that inhibit GSK-3b through virtual screening,” Bioorg Med Chem Lett, 2009, 19:533-537; Koresawa, et al., “High-throughput screening with quantitation of ATP consumption: A universal non-radioisotope, homogeneous assay for protein kinase,” Assay Drug Dev Technol, 2004, 2:153-160) (ROCK2 IC50 40 nM) (FIG. 2) and also inhibited ROCK1 with an IC50 of 76 nM (FIG. 2). In light of its potency and preliminary kinase-selectivity profile (Aurora-A IC50 values>100000 nM) (FIG. 2), CID5056270 was chosen as a starting point for the design of a focused library of aminothiazole-based small molecules as ROCK1 inhibitors. Chemical modifications of CID5056270 to improve potency, selectivity, and determine the structural features responsible for the activity, led to the identification of the urea analog 1aa (ROCK1 IC50 170 nM, ROCK2 IC50 50 nM, FIG. 2) as a novel and potent inhibitor of ROCK1.